Methionine sulfoxide reductases (MsrA and MsrB) are repair enzymes that reduce methionine sulfoxide (Met(O)) residues in proteins to methionine (Met) in a stereospecific manner. These enzymes protect cells from oxidative stress and have been implicated in delaying the aging process and progression of neurodegenerative diseases. Mammals possess one MsrA and three MsrBs. The major mammalian MsrB, MsrB1, is a selenocysteine-containing protein, whose expression and activity can be regulated by dietary selenium. Little is known about Met oxidation and repair in vivo and about identities of molecular targets of methionine sulfoxide reductases. We propose to address deficiencies in our understanding of Met(O) formation and reduction and its relevance to aging by functionally characterizing MsrA and MsrB and determining their potential for extending lifespan in animals. Specific Aim 1 is to characterize the pathway of Met(O) formation and reduction and biological functions of MsrA and MsrBs. We will determine spatial and temporal characteristics of Met oxidation and repair, identify proteins and Met residues in these proteins that are oxidized by reactive oxygen species and repaired by MsrA and MsrBs, and determine the contributions of MsrA and MsrBs to providing Met for mammalian cells. For this purpose, we will prepare new molecular tools to monitor Met(O) and will utilize them in cellular and organismal systems with altered MsrA and MsrB levels, selenium status, susceptibility to oxidative stress, and with different age of animals. Specific Aim 2 is to characterize the roles of methionine sulfoxide reductases in aging. Using fruit flies, we will determine which MsrB forms can extend lifespan and which factors are responsible for MsrB-dependent lifespan extension. In addition, we will examine a possibility that a combination of calorie restriction and selenium supplementation increases lifespan in mice. Our previous data and preliminary results suggest feasibility of these studies. PUBLIC HEALTH RELEVANCE: Oxidative damage to proteins is considered to be one of the major factors that lead to or accompany aging. This damage results from the oxidation of certain amino acid residues, among which methionine is notable because of its abundance and susceptibility to oxidation and reductive repair. Methionine sulfoxide reductases, MsrA and MsrB, are thioredoxin-dependent oxidoreductases that reduce the oxidized forms of methionine, methionine sulfoxides, to methionine in a stereospecific manner. These enzymes protect cells from oxidative stress and have been implicated in delaying the aging process and progression of neurodegenerative diseases. Mammals have one MsrA and three MsrBs, including MsrB1, a highly active selenocysteine- containing enzyme, whose expression and activity can be regulated by dietary selenium. We propose to address critical deficiencies in our understanding of methionine sulfoxide formation and reduction and its relevance to aging by functionally characterizing MsrA and MsrB and determining their potential for extending lifespan in animals.